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Accumulation and Changes in Composition of Collagens
in Subcutaneous Adipose Tissue After Bariatric Surgery
Yuejun Liu, Judith Aron-Wisnewsky, Geneviève Marcelin, Laurent Genser,
Gilles Le Naour, Adriana Torcivia, Brigitte Bauvois, Sandrine Bouchet,
Véronique Pelloux, Magali Sasso, et al.
To cite this version:
Yuejun Liu, Judith Aron-Wisnewsky, Geneviève Marcelin, Laurent Genser, Gilles Le Naour, et al.. Ac-cumulation and Changes in Composition of Collagens in Subcutaneous Adipose Tissue After Bariatric Surgery. Journal of Clinical Endocrinology and Metabolism, Endocrine Society, 2016, 101 (1), pp.292-303. �10.1210/jc.2015-3348�. �hal-01346558�
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Accumulation and Changes in Composition of Collagens in Subcutaneous Adipose Tissue Following Bariatric 1 Surgery 2 3 4 5
Yuejun Liu1,2,3,4, Judith Aron-Wisnewsky1,2,3,5, Geneviève Marcelin1,2,3, Laurent Genser1,2,3,6
, Gilles Le Naour7, Adriana Torcivia6, Brigitte Bauvois8, Sandrine Bouchet8, Véronique Pelloux1,2,3, Magali Sasso4, Véronique
Miette4, Joan Tordjman1,2,3, Karine Clément1,2,3,5 6
7
1. Institute of Cardiometabolism and Nutrition, ICAN, F-75013, Paris, France 8
2. INSERM, UMRS 1166, Nutriomic team 6, Paris, F-75013 France 9
3. Sorbonne Universités, UPMC Université Paris 06, UMRS 1166, F-75005, Paris, France 10
4. Echosens Research Department, Paris, France 11
5. Assistance Publique-Hôpitaux de Paris (AP-HP), Pitié-Salpêtrière Hospital, Nutrition Department, Paris, F-12
75013 France 13
6. Assistance Publique-Hôpitaux de Paris (AP-HP), Pitié-Salpêtrière Hospital, Department of Digestive and 14
Hepato-Pancreato-Biliary Surgery, Paris, F-75013 France 15
7. Assistance Publique-Hôpitaux de Paris (AP-HP), Pitié-Salpêtrière Hospital, Department of Pathology, UIMAP, 16
UPMC Université Paris 06, Paris, F-75013 France 17
8. Centre de Recherche des Cordeliers, INSERM UMRS 1138, Sorbonne Universités UPMC Paris 06, Université 18
Paris Descartes Sorbonne Paris Cité, F-75006 Paris, France 19
Abbreviated title: Adipose Tissue Remodeling during Weight Loss
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Key words: adipose tissue remodeling; collagen accumulation; fibrosis; cross-linking; weight loss
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Word counts (≤3600): 4230
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Number of figures and tables (≤6): 6
23 24
Corresponding author and the person to whom reprint requests should be addressed:
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Karine Clément, MD, PhD 26
Address: E3M building – 6th floor, 46-83 Boulevard de l’Hôpital, 75013, Paris. 27 Tel: 33(0) 1 4217 7928. 28 E-mail: karine.clement@psl.aphp.fr 29 30
Funding: This work was supported by several clinical research contracts (Assistance Publique-Hôpitaux de Paris
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CRC FIBROTA to JAW and KC and PHRC 0702 to KC) and funding from the Fondation pour la Recherche 32
Médicale (FRM DEQ20120323701), the National Agency of Research (ANR, Adipofib), the national program 33
“Investissements d’Avenir” with the reference ANR-10-IAHU-05 and CIFRE N° 2012/1180. 34
35
Disclosure summary: Y.L. received support from Echosens for her PhD program, M.S. And V.M are employees
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from Echosens. All other authors including J.AW., G.M., L.G., G.L.N, A.T., B.B., S.B., J.T., K.C. declare no 37
conflict of interest. 38
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Clinical Trial Registration Number: ClinicalTrials.gov NCT01655017
40 41
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ABSTRACT (249 words) 42
Context: Extracellular matrix (ECM) in subcutaneous adipose tissue (scAT) undergoes pathological remodeling 43
during obesity. However, its evolution during weight loss remains poorly explored. 44
Objective: To study histological, transcriptomic and physical characteristics of scAT ECM remodeling during the 45
first year of bariatric surgery (BS)-induced weight loss and their relationships with metabolic and bioclinical 46
improvements. 47
Patients and Main measures: 118 morbidly obese candidates for BS were recruited and followed during one-year 48
post-BS. ScAT surgical biopsy and needle aspiration, as well as scAT stiffness measurement were performed in 49
three sub-groups before and post-BS. 14 non-obese non-diabetic subjects served as controls. 50
Results: Significantly increased picrosirius-red stained collagen accumulation in scAT post-BS was observed along 51
with fat mass loss, despite metabolic and inflammatory improvements and undetectable changes of scAT stiffness. 52
Collagen accumulation positively associated with M2-macrophages (CD163+ cells) before BS but negatively after. 53
Expression levels of genes encoding ECM components (e.g. COL3A1, COL6A1, COL6A2, ELN), cross-linking 54
enzymes (e.g. LOX, LOXL4, TGM), metalloproteinases and their inhibitors were modified one-year post-BS. LOX 55
expression and protein were significantly decreased, and associated with decreased fat mass, as well as other cross-56
linking enzymes. Although total collagen I and VI staining decreased one-year post-BS, we found increased 57
degraded collagen I and III in scAT, suggesting increased degradation. 58
Conclusions: After BS-induced weight loss and related metabolic improvements, scAT displays major collagen 59
remodeling with an increased picrosirius-red staining that relates to increased collagen degradation and importantly 60
decreased cross-linking. These features are in agreement with adequate ECM adaptation during fat mass loss. 61
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The extracellular matrix (ECM) in subcutaneous adipose tissue (scAT) undergoes substantial pathological 63
remodeling during obesity. ECM accumulation, usually called fibrosis, is defined as an excessive deposition of 64
ECM components (mainly cross-linked collagens) and impaired degradation (1). ECM accumulation is important in 65
the regenerative step where it replaces damaged cells. However, if the damage persists, excessive ECM deposition 66
harms tissue homeostasis and function (2). In obesity, scAT ECM accumulation reduces tissue plasticity and results 67
in adipocyte dysfunction, ectopic fat storage, and metabolic disorders (1). Studies have shown the detrimental 68
consequences of ECM accumulation in obesity and their associations with comorbidities. In mice, genetic ablation 69
of MT1-MMP, a membrane anchored metalloproteinase degrading collagen I, leads to increased peri-adipocyte 70
fibrosis and severe metabolic complications such as hepatic steatosis (3). Likewise, Collagen VI accumulation in 71
obesity is associated with insulin resistance (4,5). By contrast, the absence of collagen VI in high-fat diet or ob/ob 72
mice results in uninhibited adipocyte expansion and associates with metabolic and inflammatory improvements (6). 73
In obese subjects, scAT fibrosis is increased (7,8). Moreover, higher scAT fibrosis at baseline is associated with 74
lower weight loss one year post-bariatric surgery (BS) (7,9). In addition, scAT pericellular fibrosis is associated 75
with liver fibrosis, suggesting that obesity is a profibrotic condition (9). Finally, the pericardial fat secretome was 76
also found to promote myocardial fibrosis (10). Overall, these studies underline the potential deleterious effects of 77
obesity-induced scAT ECM accumulation. 78
Mechanistically, scAT fibrosis leads to adipocyte dysfunction and fibro-inflammation through a mechano-79
transduction pathway (11). Lysyl oxidase (LOX), an important matrix fibers’ cross-linking enzyme, contributes to 80
tissue mechanical properties (12). In AT, LOX expression is up-regulated in high-fat diet or ob/ob mice. By 81
contrast, inhibition of LOX activity leads to improved metabolism and inflammation (13). In obese subjects, scAT 82
LOX expression is also increased (11). ScAT stiffness, measured non-invasively by transient elastography, 83
associates with picrosirius-red stained scAT fibrosis and altered glucose homeostasis (9). 84
ECM turnover, a crucial process during excess ECM accumulation, is predominately regulated by the 85
balance between matrix metalloproteinases (MMPs) and their endogenous tissue inhibitor of metalloproteinases 86
(TIMPs). In obesity, a new relationship between MMPs and TIMPs is established and enables tissue remodeling. 87
Enzymes (e.g. MMP-3, -9, -11, -12, -13, -16, and -24) are expressed at low level in scAT, but are rapidly up-88
regulated during obesity, which eventually favors scAT expansion (1). Weight loss represents another condition 89
that induces scAT remodeling, exhibiting by changes in expression of many ECM genes soon after BS (8,14). 90
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Some studies have shown increased ECM deposition, e.g. up-regulated collagens, particularly COL6A3, after 91
major weight loss in a long-term (14,15). However, most of these studies focused on selected collagens at 92
expression levels and did not explore the overall ECM characteristics. Furthermore, no study has yet evaluated the 93
links between scAT ECM remodeling, stiffness, and modifications in cross-linking enzymes, and improved 94
metabolic parameters after weight loss. 95
Herein, we examined fibrillar collagen accumulation, synthesis, and degradation as well as cross-linking 96
enzymes, macrophage infiltration and scAT stiffness during the first year post-BS. We also analyzed relationships 97
between ECM properties and metabolic and inflammatory parameters improvements observed post-BS. 98
99
Materials and Methods 100
Study Population 101
A total of 118 morbidly obese candidates for BS who met the recruitment criteria as described (7) were enrolled at 102
the Institute of Cardiometabolism and Nutrition (ICAN), Nutrition Department and operated in the Department of 103
Surgery, Pitié-Salpêtrière Hospital (Paris). Due to the difficulties to obtain large amount of scAT surgical 104
biopsy sample in every subject during the follow-up and the number of experiments to perform on these 105
samples, we divided our overall cohort into 3 groups according to the different scAT measurements that 106
were realized (study flowchart see Figure 1). However, subjects were part of the same prospective cohort and 107
baseline (T0) characteristics were not significantly different (Table 1). 108
Group1 subjects (n=52, age 40.1±10.2yr, female n=37 (71%), BS procedures: gastric banding (GB) n=8 (15%), 109
sleeve gastrectomy (SG) n=16 (31%), Roux-en-Y gastric bypass (RYGB) n=28 (54%)) accepted surgical scAT 110
biopsy before (T0) and 3 months (T3) and 12 months (T12) post-BS. Surgical biopsy was performed under local 111
anesthesia in peri-umbilical area as described (15,16). The collected scAT samples were used for explant 112
experiments and histological analysis. 113
Group2 (n=35, age 38.0±10.0yr, female n=24 (69%), BS procedures: GB n=3 (8%), SG n=16 (46%), RYGB 114
n=16 (46%)) underwent at T0, T3, and T12 scAT stiffness measurement (see below). A sub-group of 14 non-115
diabetic women from Group2 underwent scAT needle aspiration for RT-PCR analysis. Notably, 11 individuals with 116
stiffness measurement were also part of Group1. 117
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Group3 (n=42, age 42.9±10.5yr, female n=42 (100%), BS procedures: RYGB n=42 (100%)) underwent scAT 118
needle aspiration at T0 and T12 for microarray analysis. 119
14 non-obese non-diabetic subjects (age=41.6±14.1yr, female 29%, BMI=23.2±3.3 kg/m2), who had elective 120
abdominal programmed surgery without inflammatory diseases as described (7), were recruited as a control group. 121
Perioperative scAT biopsy samples were collected in the same location as in obese subjects. Ethical approval was 122
obtained from the Research Ethics Committee of Pitié-Salpêtrière Hospital (CPP Ile de France). Informed written 123
consent was obtained from all subjects. 124
Clinical, Anthropological and Biological Parameters 125
Body composition was evaluated by whole body fan-beam dual energy X-ray absorptiometry (DXA) scan (Hologic 126
Discovery W, Bedford, MA) as described (9). Blood samples were collected after 12-hour overnight fast at T0, T3, 127
and T12. Clinical variables were measured as described (7). Pancreatic beta-cell function (insulin secretion), insulin 128
sensitivity and resistance were estimated using Homeostatic Model Assessment – Continuous Infusion Glucose 129
Model Assessment (HOMA-CIGMA) (17) . 130
Measurement of scAT Shear Wave Speed by Transient Elastography 131
A new non-invasive medical device based on transient elastography (18), AdiposcanTM (Echosens, Paris, France), 132
was developed to measure scAT shear wave speed (SWS) associated with scAT stiffness (9,19). ScAT stiffness 133
was measured by the same operator in obese subjects (Group2) in the right peri-umbilical region at T0, T3 and T12. 134
Transcriptomic Experiments 135
ScAT samples obtained by needle aspiration at T0 and T12 (Group3) were stored at -80°C for microarray analysis. 136
Total RNA extraction, amplification, hybridization and raw data analysis were performed as described (20). The 137
complete dataset is published in the NCBI Omnibus (http://www.ncbi.nlm.nih.gov/geo/) through the series 138
accession number GSE72158. 139
RT-PCR for selected genes was performed as described (20), using total RNA extracted from scAT needle 140
aspiration in 14 non-diabetic obese women (Group2) at T0, T3 and T12. 141
Tissue Preparation and Histological Analysis of scAT 142
A piece of surgical biopsy sample was fixed and embedded in paraffin and sliced into 5µm-thick sections. Collagen 143
was stained with picrosirius-red (mainly collagen I and III) and analyzed using Calopix software (Tribvn, Châtillon, 144
France) in 36 subjects (Group1) at T0, T3 and T12 as described (9). Total collagen accumulation represents the 145
7
ratio of the stained fibrous area to the total tissue surface. Pericellular collagen accumulation (i.e. collagen 146
surrounding adipocytes) represents the ratio of the stained area in 10 random fields avoiding fibrosis bundles. 147
Adipocyte diameters were evaluated in the same 10 fields. Pericellular collagen accumulation was adjusted by 148
adipocyte size to eliminate the effects of different adipocyte sizes in measure fields. Collagen I and VI, degraded 149
collagen I, LOX and macrophages were detected by immunohistochemistry (IHC) using specific antibodies 150
(Supplemental Table 1). Total macrophages were defined as CD68+ cells and M2-macrophages as CD163+ cells. 151
Their results are expressed as the number of CD68+ or CD163+ cells related to 100 adipocytes (21). Collagen and 152
elastin structures were analyzed using confocal microscopy and second-harmonic generation (SHG) microscopy on 153
another piece of fixed scAT sample in 3 random obese subjects (Group1) as described (11). 154
ScAT Explant in vitro 155
Piece of surgical biopsy samples (Group1) was placed in a culture medium enriched in endothelial cell basal 156
medium (Promocell, Heildelberg, Germany), supplemented with 1% albumin free fatty acids (PAA Laboratoires, 157
Velizy-Villacoublay, France) and antibiotics. After 24-hour incubation at 37°C, scAT explant secretion media were 158
collected and frozen at -80°C for ELISA and zymography. The explant secretion was normalized to AT weight 159
according to the ratio of 1mL of culture medium for 0.1g scAT. 160
Protein Determination in scAT Explant 161
The concentrations of collagen III formation marker, N-proteases cleaved N-terminal propeptides of collagen III 162
(PRO-C3), and degradation marker, MMP-9-generated neoepitope fragments of collagen III (C3M), in scAT 163
explants were evaluated using two competitive ELISA kits developed by Nordic Bioscience A/S (Herlev, Denmark) 164
(22). The protein profiles of proMMP-2 and proMMP-9 were analyzed by zymography as described (23). 165
Statistical Analyses 166
Data are expressed as mean ± SD, categorical variables as numbers and percentages, and values in graphs as mean 167
± SEM. Categorical data were analyzed using Fisher’s exact test. For continuous data, repeated one-way ANOVA 168
was used to compare more than two groups and Holm-Sidak’s parametric multiple comparison for post-hoc 169
analysis; student’s t-test was used to compare two groups. K-means for longitudinal data (KmL) was used to 170
cluster scAT stiffness trajectories. For small sample size (i.e. n<30), data were first transformed by natural 171
logarithm if they did not follow a Gaussian distribution. Two-tailed p-values were considered significant below 172
8
0.05. All analyses were conducted using R software version 3.0.3 (http://www.r- project.org) and GraphPad Prism 173
6.0. 174
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Results 175
Increased Collagen Deposition in scAT during BS-induced Weight Loss 176
Using picrosirius-red staining, scAT collagen was quantified in 36-paired obese subjects (Group1) at baseline (T0) 177
and during the follow-up (T3 and T12). No significant difference in collagen accumulation was found among 178
the different BS procedures at baseline (Supplemental Table2). More abundant and thicker bundles of collagen 179
fibers traversing the scAT were observed at T3 and T12 (Figure 2A, B, C). Several parenchymal areas were filled 180
with less well-organized collagen in post-operative tissues (Figure 2C, enlarged image). A significant increase in 181
the average of total and pericellular collagen was observed at T3 and T12 (Figure 2D). As expected, adipocyte size 182
significantly decreased post-BS (Figure 2E), but this reduction was not correlated with collagen accumulation. 183
Moreover, the increase in pericellular collagen remained significant after adjustment for adipocyte size reduction. 184
Importantly, the fat mass reduction was negatively correlated with pericellular collagen accumulation (r=-0.40, 185
p<0.05). No other associations were observed between collagen accumulation and metabolic or inflammatory 186
variables except for systemic HDL-cholesterol (Supplemental Table3). Importantly, the results hold true in sub 187
group analysis in each different bariatric surgery technic data not shown). 188
189
Undetectable Changes in Tissue Stiffness despite Increased scAT Collagen Accumulation in scAT 190
Since we previously showed that collagen accumulation was associated with scAT rigidity and metabolic 191
alterations in obesity (9), we next aimed to investigate scAT stiffness changes post-BS using AdiposcanTM at T0, 192
T3 and T12 (Group2). To our surprise, despite increased collagen accumulation, no significant change in average 193
SWS was detected at T3 and T12 compared to T0 (T0: 0.90±0.29m/s, T3: 0.88±0.28m/s, T12: 0.93±0.43m/s, 194
p=0.58, Figure 2F). Using K-means for longitudinal data (KmL) to cluster scAT stiffness individual 195
trajectories, we observed 2 major clusters (A and B) of stiffness changes that suggest different profiles of 196
tissue response post-BS (Figure 2G). Cluster A (n=24) had significant lower stiffness than Cluster B (n=12) 197
at T0 (Cluster A 0.78±0.15m/s vs Cluster B 1.17±0.35m/s, Wilcoxon test p<0.05) and T12 (Cluster A 198
0.68±0.18m/s vs Cluster B 1.47±0.32m/s, Wilcoxon test p<0.05), while Cluster B displayed a V-shape profile 199
with an initial decrease and a subsequent increase. However, we did not observe significant bioclinical 200
differences at any time points that could possibly explain these trajectories (Supplemental Table 4). The 201
absence of significant change in average scAT SWS suggested that stiffness was not increased after surgery 202
10
despite increased picrosirius-red stained collagen. Therefore, this increase in collagen accumulation could be 203
considered as adaptive ECM remodeling requiring further investigation. 204
205
M2-Macrophages Associate with Collagen Accumulation in scAT 206
M2 cells, alternatively activated macrophages, are implicated in the resolution phase of inflammation and tissue 207
remodeling (24). Using IHC, M2 cells (i.e.CD163+ cells) and total macrophages (i.e.CD68+ cells) in scAT were 208
quantified in 15 obese subjects from Group1 at T0 and T12. The CD163+/CD68+ ratio increased between T0 and 209
T12 (0.38±0.20 vs. 0.78±0.58, p<0.01, Figure 3A), in agreement with a switch toward M2-macrophages during 210
weight loss and their role in tissue remodeling. At T0, a strong positive association between CD163+ cells and 211
pericellular collagen accumulation was observed (r=0.76, p<0.01, Figure 3D left panel). Although the number of 212
CD163+ cells moderately increased at T12 (6±3% vs. 9±4%, p=0.04, Figure 3B), a negative association between 213
CD163+ count and pericellular collagen accumulation was found (r=-0.65, p=0.02, Figure 3D right panel). By 214
contrast, the number of CD68+ cellsdecreased between T0 and T12 (17±8% vs. 14±7%, p=0.04, Figures 3C), but 215
was not associated with collagen deposition at T0 or T12. 216
217
Major ECM Remodeling at Transcriptomic Level after Weight Loss 218
As BS-induced weight loss is accompanied by increased collagen deposition without detectable one-year change in 219
SWS, we next characterized transcriptomic signatures of scAT at T0 and T12 in 42 women (Group3). Using a 5% 220
false-discovery rate, we detected 4236 up- and 2989 down-regulated genes (functional annotations see 221
Supplemental Figure 1). We focused our analysis on genes encoding proteins involved in ECM structural 222
components, profibrotic proteins, remodeling, and mechanotransduction. We found differential patterns of gene 223
changes (Figure 4A). Particularly, genes encoding collagen III (COL3A1), collagen VI (COL6A1, COL6A2), and 224
elastin (ELN) were significantly down-regulated, while collagen I (COL1A1) was unchanged. Collagen VI alpha 3 225
chain (COL6A3) was modestly up-regulated. Connective tissue growth factor (CTGF) and secreted phosphoprotein 226
1 (osteopontin, SPP1) were significantly down-regulated. MMP-9, TIMP1, TIMP2, and TIMP4 were also 227
significantly modified. Importantly, some genes previously shown to be stimulated by mechanical stress (11), such 228
as YAP, TEAD2, TEAD3, TEAD4, were not modified after weight loss (p>0.05). 229
11
During collagen biosynthesis, major post-translational modifications take place and are mediated by important 230
enzymes and chaperones. We found that the expression levels of most of these molecules were decreased at T12 231
(Figure 4B). Finally, we observed a significant down-regulation of genes encoding cross-linking enzymes such as 232
LOX, lysyl oxidase-like 4 (LOXL4), transglutaminase1 (TGM1), procollagen-lysine, 2-oxoglutarate 5-dioxygenase 233
2 and 3 (PLOD2 and PLOD3), suggesting that matrix fibers’ cross-linking was decreased post-BS (Figure 4A). 234
These transcriptomic analyses confirm the strong remodeling of scAT following BS and show major transcriptomic 235
modifications of enzymes involved in collagen biosynthesis, cross-linking and degradation. 236
237
Decreased Cross-linking of Matrix Fibers during Weight Loss Associates with Improved Metabolic 238
Phenotype 239
We next explored cross-linking enzymes and their associations with metabolic phenotypes. We confirmed 240
microarray data by RT-PCR and observed that LOX gene expression was significantly down-regulated at T3 and 241
T12 (Group2) (Figure 5A). This was substantiated by decreased LOX protein staining surrounding adipocytes at T3 242
and T12 (Figure 5B) using IHC. By confocal microscopy and SHG in fixed scAT samples in 3 random obese 243
subjects (Group1), we found a trend towards reduced collagen and elastin intensity at T3 (Supplemental Figure 2). 244
Elastin protein at T3 had more twisted structures (Figure 5C), suggesting that scAT might become less rigid after 245
weight loss. 246
We next examined the relationships between one-year changes in cross-linking enzyme expression and that of 247
clinical variables (i.e.T12-T0 variation) in Group3 (Figure 5D). The reduction of LOX gene expression was 248
positively associated with the reduction of BMI, fat mass (kg), adipocyte volume, serum leptin and orosomucoid. 249
Variation of LOXL1 was also associated with BMI, fat mass (kg), leptin, total- and HDL-cholesterol. Our gene 250
expression results suggest that decreased post-BS cross-linked scAT matrix fibers in link with improved weight 251
loss. 252
253
Increased Collagen Degradation during BS-induced Weight Loss 254
Our team (7) and others (4) have shown that collagen I and III are more frequently observed in fibrous bundles, 255
whereas collagen VI is surrounding adipocytes. Despite increased scAT collagen accumulation post-BS, we found 256
decreased collagen I and VI staining at T12 (Supplemental Figure 3), suggesting that increased picrosirius-red 257
12
staining may also indicate (at least partially) degraded collagen fragments. We tested this hypothesis by measuring 258
collagen fragments with immunostaining, ELISA and zymography from scAT explants. We observed increased 259
stained degraded collagen I surrounding adipocytes in scAT at T3 and T12 (Figure 5E). Accordingly, the 260
concentration of degraded collagen III (C3M) in scAT at T12 was significantly increased compared to T0 (Figure 261
5F left panel). ProMMP-9 and proMMP-2 entities at 92 kDa and 72 kDa respectively were observed (Figure 5G). 262
Despite individual variability, an increased trend of proMMP-2 at T3 in one non-diabetic obese subject and an 263
increase of proMMP-9 at T3 followed by stabilization at T12 in two others were observed. These changes in 264
proMMPs were not detected in samples from obese diabetic subjects (Figure 5G). In parallel, newly synthesized 265
collagen III (Pro-C3M) concentration was significantly decreased in obese compared to non-obese subjects and 266
showed a non-significant increase at T3, T12 (Figure 5F right panel). 267
268
DISCUSSION 269
Collagen accumulation in white AT is considered as an important pathological alteration associated with 270
several comorbidities of obesity (1,7,9). Our results provide new insights into weight-loss induced AT remodeling 271
in paired humans individuals before and one-year post-BS. Our results suggest that picrosirius-red stained 272
collagen in scAT does not always refer to “pathological collagens”, but could be a signature of extensive tissue 273
remodeling and collagen degradation following adipocyte shrinkage during weight loss along with improved 274
clinical, metabolic and inflammatory outcomes. 275
During physiological tissue repair, ECM accumulation is a key regenerative step replacing tissue debris and 276
dead cells (2). In pathological conditions, increased collagen deposition is not always synonymous with deleterious 277
fibrosis. In myocardial injury, different types of fibrosis have been reported according to the progression and 278
history of cardiomyopathies: a reactive “interstitial fibrosis” with ECM deposition in response to deleterious stimuli 279
is considered pathological. Conversely, a “replacement fibrosis” that replaces myocytes after cell damage or 280
necrosis may preserve the structural integrity of the myocardium (25,26). As we did not find any association 281
between adipocyte diameter reduction and pericellular collagen increase, we attribute this increased pericellular 282
collagen to “replacement collagen” that occurs at adipocyte shrinkage sites. This is further supported by our 283
observation of some large parenchyma areas filled with less well-organized collagen. This replacement collagen, as 284
part of the remodeling process, might be an adaptive and physiological phenomenon during weight-loss. 285
13
Cross-linking is necessary for matrix fibers maturation and stabilization (27) and contributes to increased 286
tissue stiffness. LOX is a major enzyme mediating collagen and elastin cross-linking. A relationship between LOX 287
enzymatic activity and tissue stiffness was established in colorectal cancer and indicated a pivotal role of LOX 288
associated stiffness in driving colorectal cancer progression (12,28). In obese subjects, scAT LOX gene expression 289
is increased (11). Increased perioperative scAT pericellular collagen is associated with increased tissue stiffness 290
measured by AdiposcanTM (9). Moreover, pericellular collagen leads to adipocyte constraints and stimulates genes 291
encoding mechano-sensitive, inflammatory and profibrotic proteins such as CTGF in a 3D model (11). Herein, 292
decreased LOX gene expression and protein, and increased elastin twist structure evaluated by SHG after weight 293
loss clearly suggest decreased cross-linking and relaxed fibers. 294
ScAT stiffness measured by AdiposcanTM relates to adipose tissue rigidity in severe obesity before
295
weight loss and is associated with picrosirius-red stained collagens and metabolic alterations (9). To our 296
surprise, we found increased post-BS collagen accumulation without significant change in average scAT 297
stiffness measured by AdiposcanTM despite large inter-individual variability. These results, associated with 298
improved metabolic alterations after BS, suggest that the major ECM remodeling observed after weight loss 299
might be adaptive. Two profiles of stiffness changes were observed without significant link with clinical 300
parameters, indicating that further studies are required to investigate the potential implication of different 301
scAT SWS profiles in BS outcomes in larger populations. Our results also suggest that transient elastography 302
AdiposcanTM might be more sensitive to severe cross-linked and dense fibrosis (i.e. “pathological fibrosis”) as 303
showed in liver stiffness measurements (29,30), thus explaining why AdiposcanTM fails to detect small decreases 304
in post-BS stiffness or alternatively to quantify adaptive ECM remodeling (i.e. less cross-linked and more 305
degraded collagens) not linked to pathological conditions. Therefore, AdiposcanTM might be more appropriate to 306
better stratify obese individuals before any drastic weight intervention or to non-invasively predict weight loss 307
outcomes (9), a feature which needs further study in extended cohorts. Furthermore, other scAT changes 308
occurring after weight loss might also influence tissue stiffness, such as the amount and types of lipids in adipocyte 309
or scAT vascularization. In addition, some genes involved in mechano-transduction pathway YAP/TEAD were 310
unchanged while the downstream profibrotic gene CTGF was down-regulated, suggesting again that weight loss 311
induced increased collagen deposition was not associated with pathological constraint. 312
14
The transcriptomic study performed before and one-year post-BS confirmed intense tissue remodeling. 313
These results align with other observations of deceased major ECM gene and profibrotic proteins both after short-314
term BS-induced weight loss (14) or dietary intervention (31). We, herein, suggest that increased picrosirius-red 315
staining is, at least partially, due to increased degraded collagens (collagen I, III) and eventually less newly 316
synthesized collagens (collagen III) as shown by immunohistochemistry and ELISA. Indeed, we found decreased 317
staining of specific collagens such as collagen I and VI. Importantly, we went beyond the transcriptomic results 318
obtained by McChulloch et al who observed only increased post-BS COL6A3 expression but not other collagen VI 319
alpha chains or their protein content (14). Our microarray analysis displayed different expression changes of 320
collagen VI alpha chain: COL6A1 and COL6A2 decreased whereas COL6A3 increased. It is well known that 321
transcriptomic changes of sub-type chains do not always relate to the same changes at the protein level. According 322
to our immunostaining results, we found less collagen VI surrounding adipocytes post-BS. 323
Our zymography analysis in scAT revealed the presence of proMMP-2 and/or proMMP-9 proteins in obese 324
non-diabetic subjects. ProMMPs are the inactive zymogen forms. There are growing evidences of the ability of 325
proMMP-2 and proMMP-9 to directly activate classical signaling pathways involved in cell growth, survival, 326
migration, and angiogenesis (32). In metabolically healthy obese individuals, scAT proMMP-9 zymographic 327
activity is increased, suggesting that proMMP-9 might be linked with better metabolic profile (33). The fact that 328
we did observe a difference in obese diabetic individuals seems to be in accordance with this last point, or 329
could also be due to the effect of anti-diabetic drugs. Exploring the co-expression of proMMPs and TIMPs in 330
the context of scAT remodeling and improved metabolism deserves further consideration. 331
The mechanisms leading to fibrosis synthesis and degradation at the cellular level need to be better 332
elineated in AT. AT macrophages (ATM) are triggers of fibrosis (34). We previously showed that both diet and 333
BS-induced weight loss improve inflammatory profiles despite non-negligible inter-individual variations (14,24,35). 334
Here, we observed increased CD163+/CD68+ ratio due to increased CD163+ cells and decreased CD68+cells during 335
weight loss, a profile of activated state of ATM shifted towards M2 relative to M1, as previously shown after 3 336
months post-BS (24). In addition, CD163+ cells before BS associated with pericellular collagen accumulation, 337
indicating a role in the generation of fibrosis in obese scAT. M2 cells have a complex role in tissue repair and 338
fibrosis: besides direct effects of M2 cells on promoting and suppressing collagen synthesis and fibrosis 339
development, M2 cells are inducers of Treg cells, which are implicated in fibrosis suppression and can directly 340
15
produce MMPs and TIMPs, thus controlling ECM turnover (36). The reason why we found a significant negative 341
association between CD163+ cells and collagen accumulation at T12 is unknown, but may suggest a balanced 342
involvement of several cell types during this remodeling process and warrants further exploration. A number of 343
studies have previously described changes of scAT immune cells quantified by IHC before and after weight 344
loss (14,24,35). However, due to the clinical difficulties in acquiring sufficient and repeated post-surgery 345
scAT surgical biopsy samples in obese subjects during the follow-up, it was hard to compare our IHC 346
observation to other methods such as fluorescence-activated cell sorting (FACS) for quantifying immune 347
cells infiltration. 348
Some questions remain unanswered. Our clinical study aimed at evaluating the changes in scAT ECM 349
until one year, the nadir point of post- BS weight loss in most individuals (37). The kinetic changes (amount, 350
type, cross-linking) of collagen fibers with longer duration of post-BS weight loss, stabilization, or weight regain 351
remains to evaluate. Some studies showed interesting results. For example, after two years post-BS weight 352
stabilization, ex-obese subjects still presented the same amount of picrosirius-red stained scAT fibrosis as morbidly 353
obese subjects, despite improvements in adipocyte hypertrophy and inflammation infiltration (38). However, 354
this ex-obese group was compared to an independent group of pre-BS obese individuals. Specific 355
comparision between two independent groups of patients before and after surgery might induce bias in the 356
results due to important inter-variability in AT fibrosis. Therefore, these findings should be confirmed in 357
samples from same individuals obtained before and after BS, as we herein assessed. Furthermore, the type of 358
collagens and cross-linking enzymes were not investigated. In addition, obese subjects experience periods of 359
weight fluctuations even post-BS (37) that could possibly subsequently modify their adipose tissue ECM 360
characteristics. We previously showed that 59 subjects who underwent RYGB after an initial failure of gastric 361
banding displayed significantly higher total collagen accumulation than primarily operated subjects (9), suggesting 362
again that weight fluctuations impact on ECM remodeling. Therefore, it is of interest to pursue the follow-up of 363
our obese subjects, who were already investigated at baseline and up until one year, to evaluate longer-term 364
scAT remodeling and potential relationships with BS outcomes. In addition, there is hardly any current data 365
concerning the change of visceral adipose tissue characteristics. In one human study, obese subjects 366
displayed decreased fat diameter in visceral AT as measured by ultrasound (39). In rodent models, mice who 367
underwent BS demonstrated decreased infiltration of T-lymphocytes and macrophages in visceral AT (40). 368
16
Further study in these post-BS features in humans would be of major interest. However, there are clinical 369
and ethical limitations to such explorations and the development of non-invasive (e.g. imaging) measures are 370
indispensable. 371
In conclusion, this study provides new insights into scAT adaptation during drastic weight-loss and shows 372
that increased picrosirius-red staining is a signature of tissue remodeling with increased collagen degradation and 373
less cross-linked fibers. It will be critical to follow patients during long-term weight loss and to determine the 374
impact of scAT remodeling on metabolic improvements. 375
17
Acknowledgements. We are grateful to the patients who contributed to this work and especially those who 377
accepted repeated surgical biopsies during the follow-up. We thank Valentine Lemoine for patients’ follow-up, 378
Florence Marchelli for data management, and Rohia Alili for her contribution in bio-banking. We thank Frédéric 379
Charlotte, Annette Lescot and Anne Gloaguen for scAT tissue preparation and picrosirius-red staining. We thank 380
Victoria Dubar for helping in immunohistostaining. We thank Claire Lovo, Aurélien Dauphin, and Christophe 381
Klein for performing SHG acquisition and for their help in analysis (Imaging Facilities, Institut du Cerveau et de la 382
Moelle épinière, PitiéSalpétrière, Paris, France). We thank Brandon Kayser, Institute of Cardiometabolism and 383
Nutrition (ICAN), for editorial/writing support. 384
18
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21
Legende 499
Table 1 500
Values are expressed as means ± SD (standard deviation), unless otherwise stated. hsCRP, highly sensitive C-501
reactive protein; HOMA-IR, homeostatic model assessment-insulin resistance; HOMA-B (%), beta cell function; 502
HOMA-S (%): insulin sensitivity. There are 11 common subjects in Group1 and Group2. For continuous data, 503
repeated ANOVA was used to compare three time points in Group1 and Group2, when p values of ANOVA were 504
<0.05, Holm-Sidak’s parametric multiple comparison test was used to compare each of the two time points; 505
student’s t-test was used in Group3. For categorical data, Fisher’s exact test was used. * p<0.05 when compared to 506
T0 in each group, # p<0.05 when compared to T3 in Group1 and Group2. Subjects’ baseline characteristics (T0) in 507
three groups were compared by ANOVA for quantitative data and by Fisher’s exact test for qualitative data. No 508
significant differences were found except the BS type. 509
22
Figure Legends 511
Figure 1. Study flowchart of 118 obese subjects. 512
Three groups of obese individuals were recruited for scAT exploration at baseline (T0) and during the follow-up 513
after bariatric surgery at 3 monthsT3 and 12 months (T12). There are 11 common subjects (female n=6) in Group1 514
and Group2 in whom we performed both histological analysis (P-R, IHC) and scAT stiffness measurement 515
(Stiffness). A sub-group of 14 subjects in Group 2 underwent scAT needle aspiration for RT-PCR analysis. 516
ScAT, subcutaneous adipose tissue; P-R, Picrosirius-red staining; IHC, immunohistochemistry; SHG, second 517
harmonic generation. 518
519
Figure 2. ScAT evaluation. 520
Collagen accumulation in scAT stained by picrosirius-red in one representative obese subject at baseline (T0) (A), 521
3 months (T3) (B) and 12 months (T12) (C) post-BS. Total and pericellular collagen accumulation (D) and 522
adipocyte size (E) at T0, T3 and T12 in 36 obese subjects from Group1. Repeated ANOVA test and Holm-Sidak’s 523
parametric multiple comparison test were used, * p<0.01. F, scAT stiffness, shear wave speed (SWS), was 524
evaluated at T0, T3 and T12 post-BS measured by transient elastography in 35 subjects (Group2). Repeated 525
ANOVA test, p > 0.05. G, scAT stiffness trajectories are clustered by K-means for longitudinal data (KmL), 526
two major clusters (A and B) of stiffness change are observed. No significant difference of stiffness at T3 was 527
observed between these two clusters (Cluster A 0.89±0.33 vs. cluster B 0.86±0.15, Wilcoxon test p=0.63) 528
529 530
Figure 3 Macrophage infiltration in scAT 531
Evolution of CD163+/CD68+ ratio (A) and CD163+ cells (B) and CD68+ cells (C) in scAT evaluated by 532
immunohistochemistry (IHC) at baseline (T0) and 12 months post-BS (T12) in 15 obese subjects from Group1 533
(IHC T0-T3-T12 sub-group). Solid lines represent non-diabetic subjects (n=8), dotted lines represent type-2 534
diabetic subjects (n=7). Pearson’s correlation between CD163+ cells and pericellular collagen accumulation at 535
baseline (T0) (D left panel) and 12months after BS (T12) (D right panel), hollow points represent Type-2 diabetic 536
subjects. 537
23
Figure 4. Transcriptomic signature of scAT ECM genes in obese subjects one year after BS 539
Gene expression levels from micro-array data in scAT at baseline represented as dotted line (T0) and 12 months 540
post-BS (T12) represented as bars, in 42 women from Group3: A, genes involved in ECM remodeling (matrix 541
fibers, cross linking, profibrotic protein, degradation proteins and adhesion protein) show important changes. B, 542
most genes involved in post-transcriptional modifications of collagen are down regulated one year post-BS. They 543
include i) enzymes involved in the hydroxylation of proline: prolyl 4-hydroxylase; prolyl-3 hydrolase;ii) enzyme 544
involved in glycosylation of hydroxylysine: GLT25D1;iii) chaperon molecules HSP47, GRP94, calexin (CANX) 545
and disulphideisomerase (PDI) (HSPA5, DNAJC10, ERP29, PDIA4, PDIA6) and iv) enzymes involved in N- and 546
C- propeptides of procollagens: ADAMTS1, ADAMTS2, ADAMTS5, ADAMTSL4. By contrast, prolyl-3 547
hydrolase (P3H2, P3H3) and ADAMTS9 genes were up-regulated. Data are presented as changes from baseline. 548
*p<0.05. 549
550
Figure 5. Cross-linking of Matrix Fibers and collagen degradation and synthesis in scAT. 551
A, Lysyl oxidase (LOX) gene expression levels at baseline (T0), 3 months (T3) and 12 months (T12) post-BS in 14 552
obese non-diabetic women from Group2. B, LOX stained by immunohistochemistry in obese and non-obese 553
subjects, X20. C, scAT elastin structure (magenta) was observed by second harmonic generation at T0 and T3 in 554
one representative obese subject among the three, X20. D, correlation heatmap between changes of bioclinical 555
parameters and changes of genes regulating cross-linking from T0 to T12 in 42 women from Group3. Correlations 556
between gene expression and changes of HbA1c and glycemia were analyzed separately in non-diabetic (nonDM, 557
n=28) and Type-2 diabetic (DM, n=14) subjects. HOMA-IR, HOMA-B% and HOMA-S% were only analyzed in 558
non-diabetic subjects. Pearson’s coefficients of each correlation are represented as blue (negative correlation) or 559
red (positive correlation), * p<0.05. E, degraded collagen I in scAT stained by immunohistochemistry in one 560
representative obese subject at T0, T3 and T12 and one representative non-obese subject. F, degraded collagen III 561
(left panel) and newly synthesized collagen III (right panel) in scAT explant measured by ELISA in 5 non obese 562
(Non Ob) and 10 obese subjects (Group1). Diabetic subjects are in red, * p<0.05. G, analysis of (pro)MMP-2 and 563
(pro)MMP-9 presence in scAT by gelatin zymography in 3 obese non-diabetic (Ob), 3 obese diabetic (Ob Diab) 564
and 2 non-obese subjects. Ob Diab 1 was under metformin at T0, but not treated at T3, T12; Ob Diab 2 was 565
under sitagliptin, glimepiride, metaformin at T0, metformin at T3, not treated at T12; Ob Diab 3 were under 566
24
insulin, liraglutide, glimepiride and metformin at T0, insulin, metformin at T3, glimepiride and metformin 567
at T12. U937 cells (ATCC CRL-1593.2) stimulated with 100 U/ml recombinant TNF for 48 h were used as 568
positive control. ProMMP-2 (72kDa) and proMMP-9 (92 kDa) were detected as transparent bands on the 569
background of Eza-blue stained gelatin. 570
Group 1 (n=52) Group 2 (n=35) Group 3 (n=42) P-R, IHC T0 T3 T12 Explant T0 T3 T12 scAT exploration SHG T0 T3 RT-PCR T0 T3 T12 Microarray T0 T12 n=36 n=13 n=3 14 n=42 n=35 Stiffness T0 T3 T12 11
A B C
D E
D
A
A B C D Ob T0 Ob T3 Ob T12 Non Ob T0 Ob T0 Ob T3 E Ob T0 Ob T3 Ob T12 Non Ob T0 F G
A
A
B
